Hcp antiserum validation using a non -interfering protein stain

Active Publication Date: 2016-10-06
BIO RAD LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes an improved method for validating polyclonal antibody mixtures used for detection of contaminating host cell proteins (HCPs) in biological samples. The method involves separating HCPs from other proteins and membrane-binding them using a non-interfering total protein stain. The non-interfering total protein stain is then removed from the membrane and the membrane is contacted with a polyclonal antibody mixture. The antibodies bind to the membrane-bound HCPs and the positions of the HCPs are detected using the non-interfering total protein stain. The non-interfering total protein stain is then removed and the membrane is contacted with a secondary detection reagent to detect the bound antibodies. The method allows for accurate validation of the polyclonal antibody mixture for detection of HCPs in biological samples.

Problems solved by technology

HCP contaminants can have an undesirable effect even at very low concentration.
For example, they can interfere with the function of the biologic.
However, the assignment of a match can be complicated when the images, or individual features within the images, are not in registry with each other.
Lack of registry between the gel image and the blot image, or features therein, can occur because of inconsistencies in electrophoresis, transfer, and staining, and the images can be of different size due to shrinkage or swelling of the gel.
These sources of inconsistency, and others, can cause incorrect or ambiguous matching.

Method used

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  • Hcp antiserum validation using a non -interfering protein stain
  • Hcp antiserum validation using a non -interfering protein stain
  • Hcp antiserum validation using a non -interfering protein stain

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Introduction

[0078]Biologic drugs, due to their origin / expression in genetically engineered host cells, their underlying physico-chemical properties, and the elaborate purification processes employed in their production, are subject to unique regulatory and technical requirements.

[0079]One of these requirements is the accurate monitoring and effective removal of process-derived impurities such as host cell proteins (HCP), host cell-derived DNA / RNA, viruses, cell culture media, chromatographic leachates, etc. (1). Among the different impurities, accurate monitoring of HCPs is perhaps the most challenging given that the expression system's proteome consists of thousands of different proteins, some of which can copurify with the biologic drug. The HCP monitoring method therefore, must be a multianalyte assay, with the ability to detect a great majority of the protein impurities that could be present in any batch of drug substance (2, 3).

[0080]Given its high sensitivity (low ppm) and its...

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Abstract

Methods and compositions are provided for validating immunological detection reagents for use in detecting contaminating host cell components in a biological preparation.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims priority to U.S. Provisional Application No. 61 / 903,234, filed Nov. 12, 2013, the contents of which are hereby incorporated by reference in the entirety for all purposes.BACKGROUND OF THE INVENTION[0002]Biologic drugs, or biologics, such as therapeutic proteins are generally derived from a host cell. For example, a host cell can be genetically engineered to produce a therapeutic recombinant protein. As another example, the biologic can be an endogenous non-recombinant protein. The host cell can be harvested and lysed, and the biologic can be purified from contaminating host cell constituents through a variety of processes. Alternately, the biologic can be secreted by the host cell into conditioned media, the media harvested, and the biologic purified from contaminants in the conditioned media. Host cell contaminants include host cell proteins (HCP).[0003]HCP contaminants can have an undesirable effect even at very l...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/15G06T7/00G01N1/30
CPCG01N33/6842G01N1/30G01N2001/302G06T7/003G01N33/15G06T7/337G01N33/6803
InventorBERKELMAN, TOM
OwnerBIO RAD LAB INC